Polyethylene glycol based oligomers for coating nanoparticles, nanoparticles coated therewith, and related methods

ABSTRACT

In a composition aspect of the invention, a nanoparticle coating comprises repeating polyacrylic acid monomers covalently bound together in an aliphatic chain having a plurality of carboxylic acid functional groups and modified carboxylic acid functional groups extending therefrom. A first portion of the modified carboxylic acid functional groups are modified by a PEG oligomer having a terminal methoxy functional group and a second portion of the modified carboxylic acid functional groups are modified by a PEG oligomer having at least one terminal catechol group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 13/608,119 filedSep. 10, 2012 and titled “Multidentate Polyethylene Glycol BasedOligomers, Nanoparticles Coated Therewith, and Related Methods,” whichclaims the benefit provisional application Ser. No. 61/532,756 filedSep. 9, 2011 and titled “Ligands for Biocompatible Nanoparticles.” Bothof these applications are incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to the field of nanoparticle coatings. Moreparticularly, the invention relates to bio-compatible nanoparticlecoatings.

BACKGROUND

In the last two decades, a variety of inorganic nanocrystals have beendesigned, synthesized and characterized, with the ultimate goals ofdeveloping a fundamental understanding of some of their unique chemical,physical and optical properties while exploiting the potential theyoffer in applications ranging from electronic devises to biology.¹⁻⁷Their properties often exhibit size and composition dependence, and arenot shared by their bulk parent material or at the molecular scale.⁴⁻⁷

These unique and controllable properties have permitted researchersacross different fields to overcome some of the limitations encounteredby conventional (bulk and such) materials for expanding old anddeveloping new technologies. Among these nanostructured materials, pureand metal-doped iron oxide nanocrystals constitute one of the mostexciting platforms due to their size-and composition-dependent magneticproperties. They have generated a great of deal of interest for use asmagnetic resonance imaging (MRI) contrast agents, in magnetic guidanceand/or separation, and as biological platforms for intracellularimaging.⁷⁻¹⁰

In the early stage of development (late 1980s), large superparamagneticiron oxide nanoparticles (SPIO, with dimensions >100 nm) containingseveral Fe₃O₄ nanocrystals were developed as in vivo T₂ MRI contrastagents.⁹ More recently, and thanks to some remarkable improvements inthe synthesis of high-quality nanocrystals using high temperature growthmethods, preparation of several iron oxide-based nanocrystals, withdemonstrated control over size-and composition-dependent magneticproperties have been reported.¹¹⁻¹³

This control has intensified interest in further enhancing the contrastefficiency and understanding the biological distribution of thesematerials inside organisms.^(13,14) However, issues of biologicaltargeting, biodistribution and in vivo toxicity of nanomaterials, ingeneral, greatly depend on their stability in complex biological media,their biocompatibility, and their hydrodynamic dimensions. Theseproperties are directly controlled by one's ability to interface themeffectively and reproducibly with biological systems.

Any nanoparticle platform with potential for use in biomedicalapplications should satisfy a few requirements, namely: 1) the surfacecoating of the nanoparticle should promote biocompatibility and reducenon-specific interactions while maintaining a compact size; 2) thenanoparticle should exhibit long-term stability in the presence of highelectrolyte concentrations and over a broad pH range; and 3) thenanoparticles should have effective and controllable surfacefunctionalization, which permits control over the number and nature ofbiomolecules attached to the nanoparticles, thus facilitating their usein applications such as targeting, sensing, and imaging.

The most effective synthetic strategies for obtaining high qualitymagnetic nanocrystals are based on a high temperature reaction oforganometallic precursors. These strategies provide nanocrystals thatare dispersible mainly in hydrophobic solutions, i.e., water-immisciblenanoparticles. Thus, additional processing using surface ligand exchangeor encapsulation within phospholipid micelles or block copolymers isrequired to transfer these materials to buffer media and to impartbiocompatibility. For instance, cap exchange with bifunctionalhydrophilic ligands is simple to implement and can produce compacthydrophilic platforms.¹³⁻¹⁸ Nonetheless, these strategies often rely onthe use of commercially available but ineffective ligands or large massblock copolymers. These approaches provide nanoparticles with limitedlong term stability and/or substantially increased hydrodynamic size.

It has been demonstrated that catechol derivatives such as theneurotransmitter dopamine and L-3,4-dihydroxyphenylalanine (L-DOPA), aprecursor to dopamine that is also used as a component of adhesivesgenerated by marine mussels, exhibit strong affinity to metal oxidenanocrystals.^(16,19,20) Several recent studies have reported thatcatechol-appended single chain PEGs provide effective capping ligandsfor iron oxide nanocrystals and permit their transfer to aqueous media.Catechol-PEG-capped iron oxide nanoparticles also have been used incellular labeling and targeted MR imaging studies^(17,18,21-23).

Although these coating polymers are water dispersible, improvement isneeded.

SUMMARY

We have developed nanoparticle coatings that are water dispersible, havea strong affinity for binding to magnetic nanoparticles, and can beeasily modified for attaching the coating to biological materials. Thenanoparticle coatings comprise a polyacrylic acid based backbone ontowhich PEG-based oligomers are appended by modifying the native carboxylgroups of the PAA backbone. The PEG-based oligomers include functionalgroups on their terminal ends, which are chosen to provide a certainfunction. Some of the terminal functional groups bind the coatings tothe nanoparticle's surface, while others provide reactive sites forbinding other compounds to the coating. The method we developed formaking these coatings allows one to tune the number and type ofPEG-based oligomers appended to the PAA backbone based on the desiredproperties of the coating.

In accordance with a composition aspect of the invention, thenanoparticle coatings comprise repeating polyacrylic acid monomerscovalently bound together in an aliphatic chain having a plurality ofcarboxylic acid functional groups and modified carboxylic acidfunctional groups extending therefrom. A first portion of the modifiedcarboxylic acid functional groups are modified by a PEG oligomer havinga terminal methoxy functional group and a second portion of the modifiedcarboxylic acid functional groups are modified by a PEG oligomer havingat least one terminal catechol group.

These and other aspects, embodiments, and features of the invention willbe better understood in the context of the accompanying drawings and thefollowing Detailed Description of Preferred Embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic diagram of the chemical structures and syntheticstrategy of making nanoparticle coatings in accordance with anembodiment of the invention. The top panel shows a preferred process formaking Compound 1. The bottom left panel shows a preferred process formaking Compound 2. The bottom right panel shows the structures ofmono-PEG-Dopa (Compound 3) and mono-PEG-COOH (Compound 4).

FIG. 2A shows ¹H NMR spectra of Compounds 1 and 2 measured in DMSO-d6.The signature of PEG around 3.26-3.64 ppm and the singlet peak from theprotons of methoxy at 3.23 ppm are dominant in spectra. Multiplet peaksaround 1.86-2.23 ppm (α) and at 6.32-6.78 ppm (*) were ascribed to theα-hydrogens of PAA and the protons in the catechols, respectively. TheInset shows a ¹H NMR spectrum of PAA.

FIG. 2B shows ¹H NMR spectra of Compounds 1-1 and 2-1 measured inDMSO-d6. The signature of azide (—CH₂N₃) at 3.39 ppm and the singletpeak from the protons of methoxy at 3.23 ppm are present in both spectra(inset). The peak signatures of PEG, the α-hydrogens of PAA, and theprotons in the catechols are identical. The peak in the spectra at 2.1ppm, marked by †, is attributed to acetone.

FIG. 3 shows TEM images of Fe₃O₄ NPs with 11, 17 and 23 nm core sizebefore (left) and after (right) ligand exchange with Compound 2 and aschematic representation of the NP with the corresponding surface capalong with images of the organic and aqueous dispersions.

FIG. 4A shows stability testing data of Fe₃O₄ NP dispersion in variouspH buffers. Shown are Mono-PEG-Dopa-stabilized (compound 3) NPs (left),OligoPEG-Dopa-stabilized (Compound 2) NPs (middle), andOligoPEG-COOH-stabilized (Compound 1) NPs (right).

FIG. 4B shows stability testing data of Fe₃O₄ NP dispersion in 1 M NaCl.Shown are Mono-PEG-Dopa-NPs (Compound 3) (left), OligoPEG-Dopa-NPs(Compound 2) (middle), and OligoPEG-COOH—NPs (Compound 1) (right).

FIG. 5A shows fluorescent dye conjugation and MR imaging data of CuAACconjugation of alkyne-functionalized dyes to azide-functionalized ironoxide NPs.

FIG. 5B shows absorption (black) and fluorescence (red) spectra afterrhodamine B conjugation of azide-functionalized OligoPEG-Dopa-NPs. TheInset shows the deconvoluted spectrum of rhodamine B.

FIG. 5C shows the T2-weighted spin-echo images (TE/TR=16/5000 ms) of thedifferent size nanoparticles with increased dilutions as shown in theinset (top) and T2* weighted gradient recalled echo images (TE/TR=3/5000ms) (bottom). The corresponding iron concentrations (in mM) in eachcapsule are shown in the sketch.

FIG. 5D shows a plot of the relative viability of Bv2 cells forincreasing concentration of OligoPEG-Dopa-stabilized Fe₃O₄ nanoparticlesperformed using an MTT assay.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the Summary above and in the Detailed Description of PreferredEmbodiments, reference is made to particular features (including methodsteps) of the invention. Where a particular feature is disclosed in thecontext of a particular aspect or embodiment of the invention, thatfeature can also be used, to the extent possible, in combination withand/or in the context of other particular aspects and embodiments of theinvention, and in the invention generally.

The term “comprises” is used herein to mean that other features,ingredients, steps, etc. are optionally present. When reference is madeherein to a method comprising two or more defined steps, the steps canbe carried in any order or simultaneously (except where the contextexcludes that possibility), and the method can include one or more stepswhich are carried out before any of the defined steps, between two ofthe defined steps, or after all of the defined steps (except where thecontext excludes that possibility).

This invention may be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will convey preferredembodiments of the invention to those skilled in the art.

The inventors have developed new set of PEG-based oligomer nanoparticlecoating ligands that have increased coordination to magneticnanoparticle surfaces, affinity to aqueous media, and the ability to beconjugated to bio-molecules. For this, the inventors have used PAA as acentral backbone onto which PEG-based oligomers are bound using a simpleapproach based on N,N-dicyclohexylcarbodiimide (DCC) coupling. ThesePEG-based oligomers can be used for coating magnetic nanoparticles suchas those comprising iron oxide

The process for making these PEG-based oligomer coatings, also developedby the inventors, allows specific functional groups such as azides,amines, and catechols to be incorporated into the coating. ThesePEG-based oligomer coatings exhibit one or more of the followingadvantageous features: (i) they include multiple nanoparticle anchoringgroups bound to a single PAA oligomer, (ii) they include multiple PEGoligomers bound to a single PAA oligomer, and (iii) the number ofreactive functional groups incorporated into the coating is tunable.

A first composition aspect of the invention is now described withreference to FIG. 1. In FIG. 1, several of the preferred PEG oligomersand nanoparticle coatings are shown. For ease of reference, each isassigned a corresponding compound number, which appears below thecompound's molecular diagram. The abbreviations used are summarized inTable 1.

In a preferred embodiment of the first composition aspect of theinvention, the PEG-based nanoparticle coating is composed of repeatingpolyacrylic acid monomer units covalently bound together to form analiphatic chain having a plurality of carboxylic acid functional groupsand modified carboxylic acid functional groups extending therefrom. Thenumber of polyacrylic acid monomer units is preferably between about 20to about 30. More preferably, the number of polyacrylic acid monomerunits is about 25.

The modified carboxylic acid functional groups are functional groupsformed by modifying a portion of the native carboxylic acid groups onpolyacrylic acid monomer units. Preferably, the modified carboxylic acidfunctional groups include amide groups formed from the combination ofthe aliphatic PAA chain with aminated PEG-based olgomers.

A first portion of the modified carboxylic acid functional groups aremodified by a PEG oligomer having a terminal methoxy functional groupsuch as —NH-PEG-OCH₃. A preferred average molecular weight for the PEGsection of this oligomer is about 750 (PEG 750). The terminal methoxyfunctional groups are advantageous because they are generally notreactive with the surface of nanoparticles or bio-molecules.

A second portion of the modified carboxylic acid functional groups aremodified by a functional group having a terminal catechol moiety.Because the terminal catechol moiety(ies) bound to the surface of ironoxide nanoparticles, they are effective at anchoring the nanoparticlecoating to the surface of the nanoparticle. Preferred catechol moietiesare formed from catechol moieties including, but not limited to—NH—R-catechol and dopamine. Here R represents an alkane, alkene, oralkyne.

Optionally, a third portion of the modified carboxylic acid functionalgroups are modified by a PEG oligomer having a terminal functional groupthat includes a terminal amine or azide moiety such as —NH-PEG-N₃. Apreferred average molecular weight for the PEG section of this oligomeris about 600 (PEG 600). compound 2, shown in FIG. 1 provides arepresentative example of nanoparticle coating having a PAA backbonehaving carboxylic acid groups modified with —NH-PEG-OCH₃,—NH—R-catechol, and —NH-PEG-N₃.

In a second composition aspect of the invention, magnetic nanoparticlesare coated with one or more of the nanoparticle coatings describedabove. The nanoparticles are preferably made of iron oxide or anothermaterial to which catechols will bind. The nanoparticles are coated bybeing directly bound to the terminal catechol moieties.

A preferred method of making multidentate polyethylene glycol(PEG)-based oligomer nanoparticle coatings, in accordance with a methodaspect of the invention, is now described.

The nanoparticle coating compositions described above are formed usingOligo-PEG-COOH precursors such as compound 1 and compound 1-1 forexample.

A first Oligo-PEG-COOH precursor is synthesized by cooling a firstsolution of PAA and a solvent to a first temperature. Preferably, thefirst temperature is approximately −5° C. to approximately 5° C., or,more preferably, about 0° C. The solvent is preferably a polar aproticorganic solvent such as THF or the like. DCC is then blended with thefirst solution to form a second solution. DMAP and the PEG oligomerhaving a terminal methoxy functional group are added to the secondsolution. The first Oligo-PEG-COOH precursor is then removed from thereaction mixture. Compound 1 is an example of the first precursor.

A second Oligo-PEG-COOH precursor is synthesized by cooling a firstsolution of PAA and a solvent to a first temperature. Preferably, thefirst temperature is approximately −5° C. to approximately 5° C., or,more preferably, about 0° C. The solvent is preferably a polar aproticorganic solvent such as THF or the like. DCC is then blended with thefirst solution to form a second solution. DMAP, the PEG oligomer havinga terminal methoxy functional group and a PEG oligomer having a terminalazide group are added to the second solution. The second Oligo-PEG-COOHprecursor is then removed from the reaction mixture. Compound 1-1 is anexample of the second precursor.

A preferred method of synthesizing a nanoparticle coating using eitherof these precursors involves preparing a first solution of EDC, DMF, andtriethylamine, then adding the desired precursor to the first solution.The catechol and DMAP are added to the first solution to form a secondsolution. The second solution is heated to between about 70° C. to about90° C. The reaction product is then purified and removed from thereaction mixture and reduced with a reducing agent such as hydrazine orthe like. The reduced reaction product is the nanoparticle coating.

A particularly advantageous feature of this method is the fact that thenumber of terminal catechol moieties, terminal methoxy groups, and azidegroups attached along the PAA chain can be engineered as desired bychanging the amount of each relative to the number of carboxyl groupsalong the native PAA chain in the synthesis process.

EXAMPLES

The embodiments of the invention described above will be even betterunderstood in the context of the following examples. These examples arenot intended to limit the scope of the invention in any way.

We have developed multidentate nanoparticle coating ligands with strongaffinity to iron oxide nanocrystals using a short PAA (Mw ˜1800 or anindex of polymerization of ˜25) as the platform/backbone to graftmultiple anchoring groups along with multiple PEG moieties within thesame structure. This design provides a small coating with a relativelylow molecular weight and accommodates several anchoring groups andseveral PEG moieties within the same structure.

We describe the use of these nanoparticle coatings to cap iron oxidenanoparticles and transfer them to buffer media. Our ligands wereprepared by laterally grafting several PEG moieties and several catecholgroups onto a polyacrylic acid short chain. In this design, theintrinsic ligand structure, including the density of anchoring groups,the size of PEG, and the type of end reactive groups can be controlled,all while maintaining a compact size. Cap exchange with these oligomerswas rapid and provided iron oxide NPs that are stable for at least 60days in the presence of large excess of added salts and over a broadrange of pHs, from pH 4 to pH 11. Colloidal stability is vastly improvedcompared to other lower coordination ligands such as mono-PEG-catecholor oligomers presenting weaker coordinating COOH groups.

We also showed that controllable fractions of azides can be introducedinto the oligomer ligand, producing NPs that are reactive withcomplementary functionalities. In particular, we demonstrated theability to couple azide-functionalized NPs to an alkyne-modified dye,which opens up the possibility of biological targeting of these NPs. Wealso measured the MRI contrast properties of these OligoPEG-capped Fe₃O₄nanoparticles and found that they exhibit strong T2 contrast enhancementwith dependence on size of the nanocrystals.

Preliminary MTT assay using these OligoPEG-NPs indicated no measurabletoxicity of the NPs to live cells. We believe that this approach can beexpanded to prepare other types of functionalized oligomers withtailor-designed anchoring groups and reactive groups, which will allowthe hydrophilic transfer and coupling of a variety of inorganicnanocrystals.

We tested nanoparticle coatings with two types of anchoring groups: (1)the native carboxyl groups present on the polyacrylic acid oligomer(compound 1) and (2) several dopamines and amine-terminated PEGs weregrafted onto the PAA, producing an oligomer that presents severalcatechol anchoring groups together with multiple PEG moieties (compound2). We also prepared and tested two molecular scale PEG-appendedligands, a mono-PEG-Dopa (compound 3) and a mono-PEG-COOH (compound 4).Here we used compounds 1, 3, and 4 as control ligands to which the dataon compounds 2 and 2-1 were compared. This allowed us to test theeffects of coordination number as well as the nature of the anchoringgroup used on the cap exchange and on the quality of the resulting Fe₃O₄nanoparticles.

FIG. 1 provides a schematic depiction of the synthetic steps involved inthe preparation of the carboxyl-and catechol-PEG-derivatized oligomers.We used commercial PAA, along with molecular scale bifunctional PEGmoieties, which we have described in previous reports.^(25,26) For theOligoPEG-COOH ligand (compound 1) a fraction of the carboxylic acidsalong the PAA backbone was reacted (via DCC coupling in THF) withNH₂—PEG-OCH₃. Characterization of the oligomer ligand using ¹H NMRspectroscopy (in DMSO-d6) showed that multiple PEG moieties were indeedcoupled to the PAA, as indicated by the appearance of a new strong broadpeak at 3.26-3.64 ppm (attributed to PEG segments) and a second sharppeak at 3.23 ppm attributed to OCH₃ groups; the weak broad peaks at1.1-2.3 ppm are ascribed to PAA (FIG. 2A). We should note that the exactlocation of these peaks depends on the solvent used. For example, a peakat 3.38 ppm was measured for this methoxy group in CDCl₃.²⁵ FT-IRanalysis further confirmed the presence of amide bonds linking the PEGmoieties to PAA with bands at 1645 cm⁻¹ and 1531 cm⁻¹. In a typicalexperiment, the degree of grafting was estimated from the ¹H NMRspectrum by comparing the relative integrations of the α-hydrogen peakfrom the acrylic acid repeat units of PAA (δ=1.86-2.23, 25H) and thethree protons in the lateral methoxy group of PEG-OCH₃ (δ=3.23, 38.8H);we measured ˜13 PEG moieties per PAA chain.

Synthesis of Azide Functionalized OligoPEG-COOH (Compound 1-1).

To prepare azide-functionalized OligoPEG-COOH (e.g., compound 1-1), amixture of OCH₃-and N₃-terminated PEG-NH₂ moieties was used during theDCC coupling reaction. By varying the relative amounts (fractions) ofazide-PEG and H₃CO-PEG moieties, one can control the number of azidegroups per oligomer. Here, we prepared and characterized of anazide-functionalized OligoPEG-COOH with a nominal azide-to-methoxy ratioof 1:3 (or 25% azide-PEG). The azide signature manifests as a tripletpeak at 3.39 ppm in the ¹H NMR spectrum; additionally, a new vibrationband at 2108 cm⁻¹ is measured in the FT-IR spectrum (FIG. 2B).

For the OligoPEG-Dopa ligands (compounds 2 and 2-1), the synthesis wascarried out in two steps: 1) First a fraction of the carboxyl groupsalong the PAA backbone was reacted (via DCC in the presence of DMAP inTHF) with either NH₂—PEG-OCH₃ or a mixture of NH₂—PEGOCH₃ andNH₂—PEG-N₃, as discussed above for the OligoPEG-COOH (compound 1 and1-1). 2) After purification, the rest of the carboxyl groups along thePEGylated oligomer intermediate were reacted (via EDC condensation) withdopamine. We found that the use of EDC condensation (instead of DCC)allowed not only a more efficient coupling between the carboxylic acidsalong the PAA (OligoPEG-COOH) and dopamine, but also facile removal ofthe byproducts and unreacted precursors by dialysis. ¹H NMR analysisconfirmed that catechol groups, PEG moieties and methoxy groups arepresent in the oligomer. In particular, a multiplet at 6.32-6.78 ppm(catechol protons), a strong peak at 3.26-3.64 ppm (due to PEG) and asharp peak at 3.23 ppm (due to methoxy) were measured in the NMR spectra(see FIG. 2). The degree of grafting was estimated to be 6.2 catecholsper chain (or oligomer), derived from comparing the relativeintegrations of the 25 α-hydrogens from the acrylic acid repeat unit ofPAA (δ=1.86-2.23, 25H) and the three protons per catechol (δ=6.32-6.78,18.5H total). In addition, FT-IR data analysis of the OligoPEG-Dopa(compound 2) showed a sizable decrease in the signature of carboxylgroups at 1722 cm⁻¹, which provides further inference that catecholgroups have been grafted onto the PAA backbone during this reactionstep.

Additional, though indirect, proof for the presence of catechol groupsin the OligoPEG-Dopa ligand structure relied on UV-Vis absorption, forwhich a clear pH-dependent change in the absorption spectra of compound2 was measured. In particular, we measured a progressive increasecoupled with broadening of the peak at 280 nm for solution of compound 2as the pH of the solution was increased. The most pronounced changeswere measured at pH≧9, with equilibrium usually reached after 90minutes. This change in absorption is similar to that measured for themono-PEG-Dopa ligand (compound 3). This change is a characteristicsignature of the catechol group in the presence of oxygen when thesolution pH is increased.

We found that ligand exchange of Fe₃O₄ nanoparticles (capped with thenative oleic acid) can be carried out using three sets of ligands,namely mono-PEG-Dopa, OligoPEG-COOH and OligoPEG-Dopa. However, weobserved substantial differences between the various ligands in the easeof implementing the cap exchange procedure and in the long termstability to changes in pH and to added electrolytes. While cap exchangewith the OligoPEG-Dopa and even the monoPEG-Dopa was rapid and requiredonly incubation at room temperature, cap exchange with OligoPEG-COOHligand required heating at ˜70° C. In comparison, mono-PEG-COOH(compound 4) could not stabilize iron oxide NPs even with longerincubation times and heating to ˜70° C. This reflects the inherent weakcoordination of a single COOH group (compared to a single catechol) toiron oxide surfaces. TEM images of nanoparticles cap exchanged withOligoPEG-Dopa (compound 2), shown in FIG. 3, indicate that the integrityof the nanoparticles was conserved, with no change in the overall sizeor shape; this confirms that the new ligands did not induce any damageor leaching of metal ions off the nanoparticle surfaces.

Side-by-side comparison of the stability of iron oxide NPs capped withthe PEGylated oligomers to pH changes showed that while both compoundsprovided dispersions that were stable over the short term, only theOligoPEG-Dopa provided well dispersed NPs over the full range of pHs.For instance, OligoPEG-COOH—NPs slowly became turbid and precipitatedafter ˜5 weeks of storage, but at pH 11 or higher precipitation occurredafter one day. Conversely, NPs cap-exchanged with mono-PEG-Dopaexhibited limited stability, as precipitation took place after 1 day atpH 4 and after 35 days at basic pH 11 or higher (see FIG. 4A). Stabilityof NP dispersions to excess electrolytes (namely 1 M NaCl) exhibited asimilar trend. The images shown in FIG. 4B indicate that OligoPEG-Dopaligand produces more stable NP dispersions than OligoPEG-COOH andmono-PEG-Dopa, as NPs capped with compound 1 precipitated after 1 monthof storage and those capped with compound 3 precipitated after 10 days.In comparison, NP capped with compound 2 stayed stable for at least 2months. When dispersed in DI water, NPs capped with compound 2 stayedstable for longer times, at least six months.

Cumulatively, these tests confirm that individually a catechol groupexhibits much stronger affinity to iron oxide than a carboxyl group,though long term stability provided by both ligands is poor. They alsoclearly prove that ligands presenting multiple carboxyl groups ormultiple catechols exhibit enhanced binding to the nanoparticle surface;sizable differences between the two types of oligomers existnonetheless. In particular, we found that the multi-catechol presentingligand (compound 2) imparts the highest stability against changes in thesolution pH and to added NaCl. This supports prior findings for whichthe benefits of multi-coordinating ligands and their ability to impartbetter colloidal stability to Au NPs and QDs (due to enhanced bindingaffinity), has been documented by us and others.^(24,25,27) However,reports on the hydrophilic stabilization of metal oxide NPs (such Fe3O4)were rather limited.^(13,21,24,28) The most promising results wereobtained mainly via encapsulation within large molecular weight blockcopolymers or silica shell, albeit with the sizable increase in the NPhydrodynamic size.²⁹⁻³¹

Our nanoparticle coatings offer a promising platform for substantiallyimproving the stability of such magnetic nanoparticles in variety ofbiologically relevant conditions, including acidic, basic, andelectrolyte-and protein-rich media.

We carried three additional and independent tests on the new set ofnanoparticles with direct implications in biology.

We first tested the ability to conjugate the newly designed OligoPEG-NPsto alkyne-modified fluorescent dye using copper(I) catalyzedazide-alkyne cycloaddition (CuAAC or Click) reaction. Fe₃O₄ NPscap-exchanged with compound 2-1 were designed to present a fewazide-terminated PEG moieties on their surfaces, making them potentiallycompatible with Click reaction. Azide-alkyne cycloaddition as a couplingstrategy has generated a tremendous interest and activity, because ofits efficiency, high chemoselectivity, and reduced crossreactivity.³²⁻³⁵ It is also compatible with a wide range of solvents andpHs. Click offers a good alternative to other more conventional reactionschemes that often rely on the reactivity of amide, ester or thioetherwith naturally abundant carboxyl, amine and thiol groups. Theirabundance, however, can induce high levels of nonspecific and unintendedconjugation of proteins and peptides.³⁵⁻³⁷

To demonstrate the compatibility of our azide-functionalizedOligoPEG-Dopa-NPs with this coupling route, we reacted the NPs with analkyne-modified rhodamine B dye (see scheme in FIG. 5A). Thedye-conjugated NPs were characterized using absorption and fluorescencemeasurements. FIG. 5B shows the absorption and fluorescence spectra ofthe dye-reacted NP dispersions following purification. Both spectraindicate the presence of bound rhodamine B onto the NPs. In particular,there is an additional absorption peak at ˜566 nm and a pronouncedfluorescence signal characteristic of the rhodamine B dye at 577 nm; thedeconvoluted dye contribution to the absorption spectrum is shown in theinsert in FIG. 5B. These optical signatures are characteristic of therhodamine B dye, clearly confirming that Click-driven coupling betweenthe azide-functionalized NPs and alkyne-rhodamine B has taken place. Theabsorption and fluorescence peaks of the NP-attached dye exhibit a smallred shift compared to the dye alone; such shifts are often observed whendyes are attached to proteins and peptides.

From the absorption data and using the extinction coefficient forrhodamine B (87,000 M⁻¹ cm⁻¹ at 554 nm, peak), we estimate that thereare ˜82 dyes per Fe₃O₄ nanoparticle. Though preliminary, this resultclearly proves the potentials of our ligand design applied to magneticNPs and could allow for developing a multimodal-imaging agent based onMR contrast.

In the second test, we evaluated the MRI contrast signature of Fe₃O₄ NPscapped with OligoPEG-Dopa ligands; (iron oxide NPs have been tested asefficient T2 MRI contrast agents). Three different size NPs (11, 17, and23 nm diameter as estimated from TEM) stabilized with compound 2 wereprepared and dispersed in buffer solutions. T1, T2 and T2* signals werecollected and used to extract estimates for r1, r2 and r2* relaxivities.FIG. 5C shows the T2 weighted spin-echo images of the different NPs withvarying dilutions, along with the corresponding T2*-weighted gradients.A strong enhancement in the T2 and T2* contrast signals withnanoparticle concentration and size was measured. We also measured aconsistent trend in the size dependence of relaxivity, with r2=181, 234,and 254 mM⁻¹ s⁻¹ for 11, 17, and 23 nm NPs, respectively.

Advantageously, the measured relaxivity r2 values for our hydrophilicFe₃O₄ NPs are well in excess of those reported for commercial clinicalagents. For example, some of the highest values reported for thoseagents (B0=0.47 T) are Feridex/Endorem (r2=160 mM⁻¹ s⁻¹), Resovist(r2=151 mM⁻¹ s⁻¹) and Sinerem (r2=160 mM⁻¹ s⁻¹).^(38,39)

Stability, biocompatibility and reduced interference with the biologicalfunctions of cells and animal functions of these magnetic nanoprobes iscritical for use in applications, such as in vitro and in vivo imagingand sensing. Our OligoPEGs present a high number of biocompatible PEGmoieties, which makes highly suitable for biological environments. Weassessed the cytotoxicity of the OligoPEG-Dopa-stabilized Fe₃O₄nanoparticles (11 nm size) using MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay ona rat microglia cell line (Bv2). The final concentration of Fe₃O₄nanoparticles in the cell media was varied between 0.0625 and 2 mM (ofequivalent Fe).

FIG. 5D shows that cell viability was largely unaffected by the presenceof the NPs. The MTT assay proves that the present set of nanoparticlesessentially has no measurable toxicity to the cells, further supportingour rationale that these multidentate PEG-rich oligomers should provideadded biological compatibility to the magnetic nanoparticles, thusenhancing their potential utility in biomedical applications.

Synthesis of OligoPEG-COOH (Compound 1).

4.0 g of poly acrylic acid (PAA, MW=1800, 2.22 mmol) was dispersed in100 mL of tetrahydrofuran (THF) in a 250 mL round bottom flask andcooled to 0° C. using an ice-bath with stirring. 4.59 g ofN,N′-dicyclohexylcarbodiimide (DCC, 22.22 mmol) was added and themixture was stirred for 30 min under N2. Then, 0.54 g of4-(N,N-dimethylamino)pyridine (DMAP, 4.44 mmol) and 16.34 g ofH₂N-PEG750-OCH₃ (22.22 mmol) were added, and the mixture was leftrefluxing for 2 days at ˜70° C. The reaction mixture was filtered usinga filter funnel and the solvent evaporated. 100 mL of deionized watercontaining 4 g of KOH were added to the residue, and the mixture wasleft stirring overnight, filtered, washed with ethyl acetate, andacidified using 1 N HCl solution to a pH 4. Chloroform was added to theaqueous solution to extract the product (3 times 100 mL each). Theorganic layers were combined dried over Na₂SO₄, filtered and the solventevaporated under vacuum to yield the crude product, which was furtherpurified using silica gel column chromatography using chloroform as theeluent.

Synthesis of Azide-Functionalized OligoPEG-COOH (Compound 1-1).

In a 250 mL round bottom flask 2.0 g of PAA (MW 1800, 1.11 mmol) weredispersed in 50 mL of THF and cooled to 0° C. using an ice-bath withstirring. 2.29 g of DCC (11.11 mmol) were added and the mixture wasstirred for 30 min under N₂. 0.27 g of DMAP (2.22 mmol), 6.13 g ofH₂N-PEG750-OCH₃ (8.33 mmol), and 1.73 g of H₂N-PEG600-N₃ (2.78 mmol)were added and the mixture left refluxing at ˜70° C. for 2 days. Thereaction mixture was filtered, the solvent was evaporated, then 100 mLof deionized water mixed with 2 g of KOH were added to the residue.After overnight stirring, the mixture was filtered then washed withethyl acetate. The aqueous phase was acidified using 1N HCl to a pH 4,and the product was extracted with chloroform (3 times 100 mL each). Thecombined organic layer was dried over Na2SO4, filtered and the solventevaporated. The product was further purified by silica gel columnchromatography using chloroform as the eluent.

Synthesis of OligoPEG-Dopa (Compound 2).

In a 100 mL round bottom flask 320 mg ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 1.67mmol) were dispersed in 10 mL of dimethylformamide (DMF) with equivalentmolar amount of triethylamine. 1 g of compound 1 (OligoPEG-COOH, 0.11mmol) was added and the mixture was cooled to 0° C. using an ice-bathwith stirring. In a separate round bottom flask (50 mL), 475 mg ofdopamine hydrochloride (2.50 mmol) was dispersed in 10 mL ofdimethylformamide (DMF) with equivalent molar amount of triethylamineand stirred for 30 min at room temperature, then 41 mg of DMAP (0.33mmol) were added. This dopamine solution was added to the solutioncontaining compound 1, and the mixture was heated to 80° C. and leftstirring for 5 days. After evaporating the solvent, 20 mL of deionizedwater containing 1 g of KOH were added to the flask, and the mixture wasstirred overnight. The mixture was filtered, washed with ethyl acetate,and after neutralization by the addition of 1 N HCl, the mixture wasdialyzed using a cellulose ester membrane with MW Cut-off of 1000 Da.The product was reduced with 1% hydrazine in deionized water, furtherpurified via 2 round of dialysis, and then lyophilized.

Synthesis of Azide-Functionalized OligoPEG-Dopa (Compound 2-1).

218 mg of EDC (1.14 mmol) was dispersed in 5 mL of DMF with equivalentmolar amount of triethylamine in a 100 mL round bottom flask. 0.66 g ofazide-functionalized OligoPEG-COOH (compound 1-1, 0.08 mmol) was addedand the mixture was cooled down to 0° C. using an ice-bath whilestirring. In a separate vial 324 mg of dopamine hydrochloride (1.71mmol) dispersed in 5 mL of DMF with equivalent molar amount oftriethylamine and the mixture was stirred at room temperature. After 30min, the dopamine solution and 28 mg of DMAP (0.23 mmol) were added tothe solution containing EDC and compound 1-1. The mixture was leftstirring at 80° C. for 5 days. After evaporating solvent, 10 mL ofdeionized water containing 0.66 g of KOH were added to the residue andstirred overnight. The solution mixture was filtered and washed withethyl acetate. After neutralization by addition of 1 N HCl solution, theproduct was purified by dialysis using cellulose ester membrane (MWCut-off=1000 Da). The product was reduced with 1% hydrazine in deionizedwater, further purified by dialysis, and lyophilized.

Cap Exchange of Iron Oxide Nanoparticles.

The iron oxide NPs were synthesized using thermal decomposition ofFe-oleate in high-boiling-point solvents, characterized and processedusing procedures developed by Hyeon and co-workers.¹² Here, we describethe procedure used for capping Fe₃O₄ nanoparticles with compound 2(OligoPEG-Dopa). 5 mg of iron oxide NPs with varying sizes (11, 17, and23 nm) dispersed in 0.5 mL of THF were mixed with 25 mg ofOligoPEG-Dopa, initially dissolved in 1 mL of ethanol, and stirredovernight at room temperature. The mixture was stirred overnight andresulted in a clear solution. The sample was then precipitated usinghexane and centrifuged to provide a dark pellet of NPs. The dark pelletwas readily dispersed in water, providing a clear solution. The aqueousdispersion was filtered through a 0.2 μm disposable syringe filter(Millipore), then excess free ligands and solubilized oleic acid wereremoved using 2 to 3 rounds of filtrations with deionized water (2-3times) using a centrifugal filtration device (Millipore, Mw Cut-off: 100kDa). Cap exchange with mono-PEG-Dopa followed the same steps as thoseemployed with compound 2. The cap exchange with OligoPEG-COOH ligand(compound 1), followed the same procedure, except that the NP/ligandmixture in THF and ethanol required a slight heating to ˜70° C. andincubation for longer times. Similar steps were employed for capping theNPs with mono-PEG-COOH.

Dye Conjugation Via Copper(I) Catalyzed Azide-Alkyne Cycloaddition.

The alkyne modified rhodamine B (RhB-alk) was synthesized via EDCcoupling between rhodamine B and propargyl alcohol following theprocedure descried in reference. Briefly, 30 μL of RhB-alk solution (2mg/ml in DMSO) was mixed with 300 μL of azide-functionalizedOligoPEG-Dopa-NPs dispersion (400 nM) in the presence of CuBr(PPh₃)₃ andstirred for 1 day at room temperature. After purification by the sizeexclusion using a PD10 column (GE healthcare) with DI water and thefiltration through a 0.4 μm disposable syringe filter (Millipore), theeluted solution was characterized using absorption and fluorescencemeasurements.

The invention has been described above with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. Unless otherwise defined, all technical and scientific termsused herein are intended to have the same meaning as commonly understoodin the art to which this invention pertains and at the time of itsfiling. Although various methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described. Theskilled should understand that the methods and materials used anddescribed are examples and may not be the only ones suitable for use inthe invention.

In the specification set forth above there have been disclosed typicalpreferred embodiments of the invention, and although specific terms areemployed, the terms are used in a descriptive sense only and not forpurposes of limitation. The invention has been described in some detail,but it will be apparent that various modifications and changes can bemade within the spirit and scope of the invention as described in theforegoing specification and as defined in the appended claims.

REFERENCES

The following references are all incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply.

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TABLE 1 Abbreviations for chemicals Abbreviation Name of chemical PAApolyacrylic acid PEG polyethylene glycol DOPA dopamine DMAP(4-(N,N-dimethylamino) pyridine) DCC N,N′-dicyclohexylcarbodiimide DMFdimethyl formamide EDC 3-dimethylaminopropyl-N′- ethylcarbodiimide DMSODimethyl sulfoxide THF tetrahydrofuran

That which is claimed is:
 1. A composition comprising repeatingpolyacrylic acid monomer units covalently bound together in an aliphaticchain having a plurality of carboxylic acid functional groups andmodified carboxylic acid functional groups extending therefrom, whereina first portion of the modified carboxylic acid functional groups aremodified by a PEG oligomer having a terminal methoxy functional groupand a second portion of the modified carboxylic acid functional groupsare modified by a PEG oligomer having at least one terminal catecholgroup.
 2. The composition of claim 1, wherein the PEG oligomer having aterminal methoxy functional group comprises a PEG chain having aterminal amine group opposite the terminal methoxy functional group, theterminal amine group being covalently bound to a carbonyl carbon of amodified carboxylic acid functional group.
 3. The composition of claim1, wherein the PEG oligomer having at least one terminal catechol groupis dopamine.
 4. The composition of claim 1, further comprising a thirdportion of modified carboxylic acid functional groups wherein the thirdportion of the modified carboxylic acid functional groups are modifiedby a PEG oligomer having functional group.
 5. The composition of claim1, wherein the number of polyacrylic acid monomer units is between about20 to about 30.